Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ibα using canine–human chimeras

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Numerous studies have implicated bacteria in cardiovascular disease, but there is a paucity of information on the mechanism involved. In this study we show how the common oral bacterium Streptococcus sanguis can directly interact with platelets, resulting in activation and aggregate formation. Platelet aggregation was dependent on glycoprotein IIb/IIIa (GPIIb/ IIIa) and thromboxane. Platelets could also directly bind to S sanguis, but this interaction was not inhibited by GPIIb/IIIa antagonists. Antibodies to GPIb could inhibit both platelet aggregation and platelet adhesion to bacteria. This suggested a direct interaction between GPIb and S sanguis; however, this interaction did not require von Willebrand factor, the normal ligand for GPIb. By use of a range of monoclonal antibodies to GPIb and the enzyme mocharagin, which cleaves GPIb at amino acid 282, the interaction was localized to a region within the N-terminal 1-225 portion of GPIb␣. Furthermore S sanguis failed to induce aggregation of platelets from a patient with BernardSoulier disease, the organism bound to Chinese hamster ovary cells transfected with the GPIb␣ gene but did not bind to mock-transfected cells and biotin-labeled S sanguis cells bound to purified GPIb in ligand blots. It is suggested that the interaction between S sanguis and GPIb is important in the pathogenesis of infective endocarditis and may also play a contributory role in some cases of myocardial infarction. ( IntroductionRecent reports suggest a role for infectious agents in cardiovascular disease. Much of this work is in the form of clinical evidence of infection [1][2][3][4][5][6] or the effect of antibiotics on the incidence of cardiovascular disease. 7,8 Although studies have found evidence of bacteria in atherosclerotic plaques, 1,2,4,5,9-13 their role in the etiology of cardiovascular disease is uncertain. In contrast, the role of bacteria in infective endocarditis is well established and the molecular mechanisms involved may also occur in other forms of cardiovascular disease.Infective endocarditis involves inflammation of the heart valves due to infection and if untreated can lead to valve failure and death. In most cases there is one or more predisposing factors, which results in damage to the endothelium on or adjacent to the valves. This area of damage becomes covered with a platelet-fibrin vegetation and these can become colonized by bacteria that gain access to the blood. The 2 species most commonly involved are Streptococcus sanguis 14 and Staphylococcus aureus. 15 Historically oral streptococci have been referred to as Streptococcus viridans, but this name was never accepted as a recognized taxon because of the biochemical and serologic heterogeneity among isolates. Subsequent detailed biochemical and genetic studies allowed the definition of at least 17 taxa within what was originally called S viridans. However, the term viridans has survived but is now used as viridans group of streptococci to recognize the existence of various taxa and S sanguis is one taxon w...

Section: Discussionmentioning

confidence: 72%

mentioning

confidence: 72%

A role for glycoprotein Ib in Streptococcus sanguis–induced platelet aggregation

Kerrigan

Douglas

Wray

et al. 2002

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144

“…27 Of all the GP Ib␣ antibodies, only AP1 can block the interaction of GP Ib␣ with VWF, thrombin, and Mac-1, rendering its epitope of intense interest for understanding the ligand-binding functions of GP Ib␣. The AP1 epitope has been narrowed to the sequence Phe201 to Glu225, 28 which comprises the ␣1 helix followed by the ␤13 strand. 3,4 In this study, our intent was to map the epitope in detail and to test the effects on VWF binding function of amino acid substitutions within the epitope.…”

Section: Introductionmentioning

confidence: 99%

The α1 helix-β13 strand spanning Leu214 to Val229 of platelet glycoprotein Ibα facilitates the interaction with von Willebrand factor: evidence from characterization of the epitope of monoclonal antibody AP1

Peng

Berndt

Crúz

et al. 2004

Self Cite

The glycoprotein Ib-IX-V (GP Ib-IX-V) complex mediates platelet binding to von Willebrand factor (VWF) through its largest polypeptide, GP Ib␣. Of the many GP Ib␣ monoclonal antibodies described, AP1 is of particular interest because it blocks static VWF binding induced by 2 modulators, ristocetin and botrocetin, and platelet adhesion to VWF surfaces under flow. We mapped the AP1 binding site to a region encompassing Arg218 to Tyr228, comprising the ␣1 helix and ␤13 strand defined by the GP Ib␣ crystal structure. AP1 binding absolutely required Arg218, Asp222, and Glu225. We evaluated the ability of cells expressing mutants of this region to bind VWF under static conditions in the presence of modulators, and to attach to and roll on a VWF matrix under flow. These data indicate that 2 regions within the sequence Arg218 to Tyr228 have important roles in VWF binding: the ␣1 helix has a regulatory role and the ␤ turn and ␤13 strand bind VWF directly. Despite this, the only effect of a synthetic peptide corresponding to Leu214 to Val229 was to slightly increase the rolling velocity of GP Ib␣-expressing Chinese hamster ovary (CHO) cells on VWF. This region thus appears to be more important for maintaining the regional conformation of GP Ib␣, thereby facilitating the interaction with VWF. (Blood. 2004; 104:3971-3978)

“…The mAb AK2 binds at the first LRR, residues 36-59. 20,21 The mAb VM16d binds at residues 201 to 268 of the C-terminal region 13,21 and WM23 binds at the macroglycopeptide region downstream to residue 282 20 ( Figure 6A). The binding of AK2 and VM16d were compared to that of WM23, which binds outside the VWF-binding region, to adjust for differences in surface expression of GPIb␣ between transfected cell lines.…”

Section: Expression Of the Wt And Mut Gpib␣ Receptor In Cho ␤/Ix Cellsmentioning

confidence: 99%

“…[8][9][10] Four regions within the amino-terminal domain contribute to VWF binding: N-terminal flank (His1-lle35), LRRs (Leu36-Ala200 residues), C-terminal disulfide loop (Phe201-Gly268), and an anionic tyrosine sulfated region (Asp269-Glu282). [11][12][13] The interaction between VWF and its platelet receptor GPIb␣ is essential for platelet adhesion at sites of high-shear stress. Structural changes affecting GPIb␣ are responsible for the PT-VWD phenotype.…”

Section: Introductionmentioning

confidence: 99%

Identification and functional characterization of a novel 27-bp deletion in the macroglycopeptide-coding region of the GPIBA gene resulting in platelet-type von Willebrand disease

Othman

Notley

Lavender

et al. 2005

Interaction between the platelet glycoprotein Ib␣ (GPIb␣) receptor and its adhesive ligand von Willebrand factor (VWF) has a critical role in the process of hemostasis. Platelet-type von Willebrand disease (PT-VWD) is a rare bleeding disorder that results from gain-of-function mutations in the GPIBA gene. We studied this gene from 5 members of a previously unreported family with a PT-VWD phenotype. We identified a novel in-frame deletion of 27 base pair (bp) in the macroglycopeptide region. This deletion was not found in the unaffected family members or in 50 healthy controls. The patients' platelets expressed normal quantities of GPIb/IX/V complex on their surface and the mutant (Mut) GPIb␣ was expressed at levels indistinguishable from the wild-type (WT) receptor on the surface of transfected Chinese hamster ovary ( IntroductionPlatelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder first described in 1982. 1 It results from an abnormally high affinity interaction between the platelet membrane glycoprotein (GP) Ib/IX/V complex and von Willebrand factor (VWF), leading to characteristic platelet hyperaggregability. 1,2 Patients with PT-VWD have frequent and severe nosebleeds and excessive bleeding following tooth extraction, tonsillectomy, and other surgical operations. 3 The condition shares most of the clinical and laboratory features of type IIB VWD and the final discrimination between them requires either platelet-mixing studies or a molecular genetic approach. Rapid clearance of highmolecular-weight (HMW) VWF multimers from plasma together with thrombocytopenia caused by an increased removal of the aggregated platelets contribute to the bleeding diathesis in PT-VWD. 3,4 The GPIb/IX/V platelet receptor comprises 4 transmembrane polypeptides, each of which belongs to the leucine-rich repeat (LRR) family of proteins. They are GPIb␣ disulfide linked to GPIb␤, GPIX, and GPV, each of which is noncovalently associated with the complex with a stoichiometry of 2:2:2:1. 5,6 The 2 proteins, GPIb␤ and GPIX, are required for efficient cell-surface expression of GPIb␣. 7 GPIb␣ is the largest component of the receptor (610 amino acids) and carries the VWF-binding site. The structure of GPIb␣ reveals 4 functional domains: the 45-kDa amino-terminal domain; a heavily glycosylated mucinlike stalk known as the macroglycopeptide region, a single transmembrane domain, and an intracytoplasmic domain. [8][9][10] Four regions within the amino-terminal domain contribute to VWF binding: N-terminal flank (His1-lle35), LRRs (Leu36-Ala200 residues), C-terminal disulfide loop (Phe201-Gly268), and an anionic tyrosine sulfated region (Asp269-Glu282). [11][12][13] The interaction between VWF and its platelet receptor GPIb␣ is essential for platelet adhesion at sites of high-shear stress. Structural changes affecting GPIb␣ are responsible for the PT-VWD phenotype. 14,15 To date, 5 families and one isolated case of PT-VWD have been reported and in these cases the responsible mutations reside within the ...